Cranial Suture Regeneration Mitigates Skull and Neurocognitive Defects in Craniosynostosis.

Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.

Cranial suture lineage and contributions to repair of the mouse skull.

The cranial sutures are proposed to be a stem cell niche, harbouring skeletal stem cells that are directly involved in development, homeostasis and healing. Like the craniofacial bones, the sutures are formed from both mesoderm and neural crest. During cranial bone repair, neural crest cells have been proposed to be key players; however, neural crest contributions to adult sutures are not well defined, and the relative importance of suture proximity is unclear. Here, we use genetic approaches to re-examine the neural crest-mesoderm boundaries in the adult mouse skull. These are combined with calvarial wounding experiments suggesting that suture proximity improves the efficiency of cranial repair. Furthermore, we demonstrate that Gli1+ and Axin2+ skeletal stem cells are present in all calvarial sutures examined. We propose that the position of the defect determines the availability of neural crest-derived progenitors, which appear to be a key element in the repair of calvarial defects.

Expansion of the sagittal suture induces proliferation of skeletal stem cells and sustains endogenous calvarial bone regeneration.

In newborn humans, and up to approximately 2 y of age, calvarial bone defects can naturally regenerate. This remarkable regeneration potential is also found in newborn mice and is absent in adult mice. Since previous studies showed that the mouse calvarial sutures are reservoirs of calvarial skeletal stem cells (cSSCs), which are the cells responsible for calvarial bone regeneration, here we hypothesized that the regenerative potential of the newborn mouse calvaria is due to a significant amount of cSSCs present in the newborn expanding sutures. Thus, we tested whether such regenerative potential can be reverse engineered in adult mice by artificially inducing an increase of the cSSCs resident within the adult calvarial sutures. First, we analyzed the cellular composition of the calvarial sutures in newborn and in older mice, up to 14-mo-old mice, showing that the sutures of the younger mice are enriched in cSSCs. Then, we demonstrated that a controlled mechanical expansion of the functionally closed sagittal sutures of adult mice induces a significant increase of the cSSCs. Finally, we showed that if a calvarial critical size bone defect is created simultaneously to the mechanical expansion of the sagittal suture, it fully regenerates without the need for additional therapeutic aids. Using a genetic blockade system, we further demonstrate that this endogenous regeneration is mediated by the canonical Wnt signaling. This study shows that controlled mechanical forces can harness the cSSCs and induce calvarial bone regeneration. Similar harnessing strategies may be used to develop novel and more effective bone regeneration autotherapies.

Cranium growth, patterning and homeostasis.

Craniofacial development requires precise spatiotemporal regulation of multiple signaling pathways that crosstalk to coordinate the growth and patterning of the skull with surrounding tissues. Recent insights into these signaling pathways and previously uncharacterized progenitor cell populations have refined our understanding of skull patterning, bone mineralization and tissue homeostasis. Here, we touch upon classical studies and recent advances with an emphasis on developmental and signaling mechanisms that regulate the osteoblast lineage for the calvaria, which forms the roof of the skull. We highlight studies that illustrate the roles of osteoprogenitor cells and cranial suture-derived stem cells for proper calvarial growth and homeostasis. We also discuss genes and signaling pathways that control suture patency and highlight how perturbing the molecular regulation of these pathways leads to craniosynostosis. Finally, we discuss the recently discovered tissue and signaling interactions that integrate skull and cerebrovascular development, and the potential implications for both cerebrospinal fluid hydrodynamics and brain waste clearance in craniosynostosis.

Onabotulinumtoxina (craniotomy scar combined with cranial suture line injections) for persistent post craniotomy headache: Case series with long-term follow-up.

BACKGROUND: There is no defined preventive treatment protocol for persistent post-craniotomy headache. In several small case series and individual case reports onabotulinumtoxinA injected into the craniotomy scar has shown possible efficacy. What is lacking is long term follow-up and if focusing on the cranial suture lines along with the craniotomy scar can enhance improvement and provide more sustained benefit. METHODS: Retrospective chart review with case series. RESULTS: Four patients (three women, one man) with ICHD-3 defined persistent post craniotomy headache were treated using a novel onabotulinumtoxinA injection protocol. All the patients presented with continuous head pain of moderate to severe intensity. All had severe allodynia on the side of their craniotomy. All had significant reduction in quality of life. Our application of onabotulinumtoxinA involved injection into both the surgical scar and the transected/irritated cranial suture lines noted on neuroimaging and physical examination. With treatment all patients demonstrated significant benefit including a reduction in daily pain intensity (75%-100%), developing periods of pain freedom (2-7 days per week) and having a dramatic improvement in quality of life (close to 100% in all). The benefit was sustained for at least five years of follow-up. CONCLUSION: From our case series it appears that injection not only along the painful craniotomy scar but into the involved cranial suture lines provides positive efficacy and sustained improvement in patients with persistent post craniotomy headache.

The biomechanics of chewing and suckling in the infant: A potential mechanism for physiologic metopic suture closure.

Craniosynostosis is a condition with neurologic and aesthetic sequelae requiring invasive surgery. Understanding its pathobiology requires familiarity with the processes underlying physiologic suture closure. Animal studies have shown that cyclical strain from chewing and suckling influences the closure of cranial vault sutures, especially the metopic, an important locus of craniosynostosis. However, there are no human data correlating strain patterns during chewing and suckling with the physiologically early closure pattern of the metopic suture. Furthermore, differences in craniofacial morphology make it challenging to directly extrapolate animal findings to humans. Eight finite-element analysis (FEA) models were built from craniofacial computer tomography (CT) scans at varying stages of metopic suture closure, including two with isolated non-syndromic metopic craniosynostosis. Muscle forces acting on the cranium during chewing and suckling were simulated using subject-specific jaw muscle cross-sectional areas. Chewing and suckling induced tension at the metopic and sagittal sutures, and compressed the coronal, lambdoid, and squamous sutures. Relative to other cranial vault sutures, the metopic suture experienced larger magnitudes of axial strain across the suture and a lower magnitude of shear strain. Strain across the metopic suture decreased during suture closure, but other sutures were unaffected. Strain patterns along the metopic suture mirrored the anterior to posterior sequence of closure: strain magnitudes were highest at the glabella and decreased posteriorly, with minima at the nasion and the anterior fontanelle. In models of physiologic suture closure, increased degree of metopic suture closure correlated with higher maximum principal strains across the frontal bone and mid-face, a strain regime not observed in models of severe metopic craniosynostosis. In summary, our work provides human evidence that bone strain patterns from chewing and suckling correlate with the physiologically early closure pattern of the metopic suture, and that deviations from physiologic strain regimes may contribute to clinically observed craniofacial dysmorphism.

Atypical sagittal suture craniosynostosis: pathological considerations for early closure of the anterior part of the sagittal suture.

Sagittal suture synostosis is one of the most common craniosynostoses and is often diagnosed by characteristic narrow and long skull shape, scaphocephaly. However, some patients with sagittal suture synostosis do not present with typical scaphocephaly, making early diagnosis difficult. In this study, five cases of characteristic skull deformity showing a narrowing of the cranium posterior to the coronal suture on computed tomography (CT) are presented. The three older children presented with papilledema and intellectual disability and a closed sagittal suture on CT. The two infant cases were diagnosed with the characteristic cranial deformities with aggravation of the deformity over time, but sagittal suture closure was not evident on CT. All patients underwent cranial remodeling surgery. In the two infant cases, the histopathological findings showed that the anterior part of the sagittal suture was firmly fused with fibrous tissue without bony fusion. These findings suggested that narrowing of the cranium posterior to the coronal suture might be due to functional fusion of the anterior portion of the sagittal suture prior to bony fusion. In an infant presenting with such a deformity that shows aggravation of the deformity over time, surgical treatment should be considered.

Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-ß signaling.

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-ß signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-ß signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-ß signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

Wormian Bones: Prevalence, Topography, and Implications.

BACKGROUND: Wormian bones also known as sutural bones are asymmetrical and shapeless bones occurring in cranial sutures and fontanelle of the skull. Their incidence is reported to vary in population. The exact etiology is debatable, but their formation is under the control of both genetic and environmental factors and has great anthropological and clinical implications related to the cranium. Due to high clinical relevance of Wormian bones, the study was carried out. The aim of the study is to expound the incidence and topography of Wormian bones along with clinical significance in dry adult skulls of Indian origin. METHODS: The study was conducted in the Department of Anatomy using 200 complete adult dry skulls of unknown age and sex. All the sutures in the skull were inspected for the presence of Wormian bones. The Wormian bones were classified into 10 categories, and associated implications were brought out. The Wormian bones were photographed, and details were compared with the available literature. RESULTS: The Wormian bones were observed in 190 (380 sides) skulls with 186 on the right side and 108 on the left side. The predominance site of sutural bone was lambdoid suture followed by sagittal suture. The least common site of Wormian bone was lambda. CONCLUSION: The detailed information of precise topography, frequency, and number of Wormian bones is of utmost use for surgeons performing surgery on the skull, anthropologists for identifying races, and forensic scientists for investigating child abuse cases.

The Accuracy of Classification Systems in Nonsyndromic Sagittal Craniosynostosis.

Numerous classification systems of nonsyndromic sagittal craniosynostosis (NSC) are applied but none has gained a wide acceptance, since each classification is focused on distinct aspects. The aim of the study was to assess the accuracy of 4 classifications of NSC discussed in the literature by defining the associations among the classifications, individual features (sex, age, cranial index), and objective morphologic criteria (frontal bossing, retrocoronal constriction, sagittal ridge, and occipital bulleting). The study was conducted on anonymized thin-cut CT scans of 133 children with NSC 1 to 12 months old (mean age 5.42 mo). The type of cranial dysmorphology was assessed using 4 classification systems, focusing on skull shape, pattern of sagittal suture closure (Heuzé classification), deformation of skull vault (Sakamoto classification), and a single-dominant feature (David classification). Each patient was also independently investigated for the presence of morphologic criteria. A multivariate analysis was performed to explore the relations among the classifications and assess their accuracy. In the analyzed cohort sphenocephaly (38.3%), CFF type by Heuzé (30.8%), type I by Sakamoto (72.9%), and a central type by David (42.9%) were dominant findings. Regarding the morphologic criteria, frontal bossing was observed the most frequently (91.7%). The age of patients and cranial index differed significantly among the shapes of skull and David classifications ( P <0.001). The shape-based system showed the strongest correlation with other classifications and with measurable variables. Other classifications have much in common and some overlap, but none of them constitutes a standalone system to define all aspects of cranial dysmorphology in NSC.

Surgical Treatment of Trigonocephaly, Simplified Technique for Moderate Cases.

BACKGROUND: The prevalence of trigonocephaly has increased worldwide over the past 2 decades. Early identification and appropriate treatment are critical. The aim of this study is to evaluate the outcomes and the effect of metopic suture excision, perisutural frontal bone shave, and bilateral pericranial flap method on the shape of the forehead after surgical correction in infants with moderate trigonocephaly. METHODS: The present study was performed as a cross-sectional study on 40 infants of 3 to 12 months old with trigonocephalus who underwent metopic suture excision and pericardial flap surgery in Mofid Pediatric Hospital from 2016 to 2022. The definitive diagnosis of patients' trigonocephaly was made based on clinical signs and computed tomography scan findings by a plastic surgeon. RESULTS: Overall in 40 patients operated by this technique, 23 (57.5%) of cases were males, and 17 (42.5%) were females. The mean age of patients was 7.86 ± 2.22 months. Hospital stay was 2 to 4 days (mean: 3 d), intensive care unit admission was in 33 cases for 24 hours, and no intensive care unit admission for 7 cases. Blood was transfused during surgery for 25 patients, and 15 patients did not require blood transfusion use. Results were evaluated in 6 to 12 months after surgery by 3 independent plastic surgeons, with pre and postoperative photos. Satisfaction with the results of forehead shape was excellent for 60% of patients, good for 37.5%, and moderate for 2.5%. Only one female patient had a recurrence after the surgery. CONCLUSION: This study showed that the pericranial flap method after full metopic suture excision and frontal shave was very effective in the treatment of infants with moderate trigonocephaly.

Evaluation of Bone Thickness and Density Values in Resistance Regions in Rapid Maxillary Expansion Using Computed Tomography.

OBJECTIVES: This study used computed tomography (CT) to compare the bone thickness and density values around the zygomaticomaxillary, zygomaticotemporal, and pterygomaxillary sutures that are resistant to rapid maxillary expansion (RME) treatment according to age, sex, and cervical vertebrae maturation (CVM) stage. METHODS: The study included 200 paranasal sinus records obtained for medical diagnosis and examination in a radiology clinic. The records provided data on 110 males and 90 females aged between 4 and 28 years. Bone thickness and density values around the zygomaticomaxillary, zygomaticotemporal, and pterygomaxillary sutures were measured using CT imaging. The correlations of bone thickness and density values with the variables of age, sex, and CVM stage were evaluated. RESULTS: No statistically significant difference was revealed between the bone thickness values around the zygomaticomaxillary and zygomaticotemporal sutures and age, sex, CVM stage, and the right and left regions of the same individual ( P >0.05). A strong correlation was identified between Hounsfield units (Hu) values on bone density in all 3 regions and age and sex ( P <0.001). No correlation was found between the CVM stage and density values around the zygomaticomaxillary, zygomaticotemporal, and pterygomaxillary sutures ( P >0.05). CONCLUSIONS: The Hu values of the records from females were higher than those of males in all age groups. It was observed that with increasing age, bone density values increased in all 3 regions, and thus circummaxillary region's Hu value increased.

Midpalatal suture maturation in 15- to 35-year-olds: morphological assessment in the coronal plane using CBCT-an exploratory study.

Rapid maxillary expansion (RME) is used in patients presenting transverse maxillary deficiency. However, RME may be unpredictable after late adolescence if the midpalatal sutural maturation stage (MPMS) is in late stage. Since MPMS evaluation is influenced by the expertise of the operator and image quality, this classification method could be complemented. Therefore, this study aimed to analyze the morphology of the midpalatal suture (MPS) and its surrounding bone in the coronal plane using cone beam computed tomography (CBCT) images of adolescents and young adults and to correlate the findings with their respective MPMS status. CBCT scans of 200 patients aged 15-35 years of both sexes were evaluated. MPS and surrounding bone tissue characteristics in the coronal and axial sections were analyzed, and MPMS was evaluated (A to E). Six categories were identified for the coronal evaluation, analyzing it in 2 standardized locations. Sutural morphology: I, hypodense sutural line limited by 2 hyper-dense para-sutural lines; II, hypodense sutural line; and III, suture not visible. The presence of para-sutural cancellous bone: CB0, compact bone; CB1, dense cancellous tissue; CB2, spaced cancellous bone. Midpalatal bone thickness (MBTh) was also measured. ANOVA-Bonferroni, chi-square, logistic regression and t-tests were used. MPMS increased with age. Sutural morphology I and CB0 bone were the most prevalent. Here we show that sutural morphology, para-sutural bone characteristics, and MPSM were significantly associated. The coronal evaluation of the MPS can complement the information from the MPSM assessment, allowing the identification of patent MPS even in the presence of late MPSM.

Occurrence of metopic suture in modern and archaeological Croatian population.

AIM: To determine the incidence of metopism in the modern and archaeological Croatian population. METHODS: A total of 800 specimens (454 modern multi-slice computed tomography [MSCT] scans and 346 dry archaeological skulls) were visually examined for metopic suture presence. The metopic suture was deemed complete when aligned nasion to bregma. RESULTS: In the overall sample, the metopic suture was observed in 36 of 800 subjects (4.5%): 19 of 424 (4.5%) men and 17 of 370 (4.6%) women. A significant difference was not observed between modern and archaeological samples (χ2=3.219, P=0.359) or between the sexes (χ2=0.006, P=0.939). The frequency of metopism varied from 3.5% in the modern population to 7.04% in the samples from the Roman period. CONCLUSION: There are no visible secular changes on metopic suture in the Croatian population through time. Some variations can be the result of differences in sample size in different time periods.

Anatomical variations of the zygomaticofacial foramen and its related canal through the zygomatico-orbital and zygomaticotemporal foramina in dry human skulls.

PURPOSE: The aim of this study is to reveal the location of the zygomaticofacial foramina, the variations of their numbers, and their connections between the zygomatico-orbital and zygomaticotemporal foramina. METHODS: Ethics committee approval of our study was received by the Istanbul Medical Faculty Clinical Research Ethics Committee (date:30.07.2021, number:358356). 171 zygomatic bones of unknown gender from the Department of Anatomy, Istanbul University, were included in this study. The number of zygomaticofacial foramina and their connections with the zygomatico-orbital foramen and the zygomaticotemporal foramina were examined. Also, the morphometric distances between the zygomaticofacial foramen were calculated. Evaluation of the data was done with SPPS v.21. RESULTS: The number of zygomaticofacial foramina was found as 299. It was found single, double, three, four, five and six foramina, in 52 (30.4%), 52 (30.4%), 24 (14.03%), 10 (5.85%), 5 (2.93%), 1 (0.58%) zygomatic bone, respectively. Zygomaticofacial foramen was absent in 27 (15.8%) bones. Of these 299 foramina, 129 were found to be connected with zygomatico-orbital foramen and 23 with zygomaticotemporal foramen. It was noted that 147 zygomaticofacial foramina had no connection with any foramina. The distances between the zygomaticofacial foramen and the frontozygomatic suture, temporal process, maxillary process, the lowest point of the zygomatic bone, and orbital rim were found as 25.30 ± 2.81mm, 18.74 ± 3.56mm, 21.56 ± 4.16mm, 18.72 ± 2.57mm, 6.67 ± 3.27mm, respectively. CONCLUSION: Consequently, the location and variations of ZFF are of great importance for maxillofacial surgery and regional block anesthesia. Knowing its location and variations will help prevent complications during any surgical intervention in this region.

Unreported variant foramen of the frontal bone: the latero-orbital foramen.

Variant foramina of the skull can lead to misdiagnosis on medical imaging and potentially, intraoperative complications if not appreciated. Here, we report an unusual foramen found superior to the frontozygomatic suture. The foramen was located on the left side at the superolateral rim of the orbit, 2.36 cm inferolateral to the supraorbital foramen. It was positioned 2.5 mm superior to the frontozygomatic suture. The foramen had a length of 3.1 mm and a width of 1.3 mm. The internal opening of the foramen was located 1.45 cm superolateral to the zygomaticotemporal foramen. We suggest that this foramen is a pathway for either a branch of the zygomatic nerve or lacrimal nerve and/or their vascular bundles. Although the prevalence of such a finding cannot be confirmed, such a case is of archival value as a comparison for future similar cases.

Performance of dental students, orthodontic residents, and orthodontists for classification of midpalatal suture maturation stages on cone-beam computed tomography scans - a preliminary study.

BACKGROUND: Assessment of midpalatal suture maturation on cone-beam computed tomography (CBCT) scans is performed by visual inspection and is therefore subjective. The extent to which the assessment of midpalatal suture maturation is affected by rater experience has not been adequately explored in the existing literature, thus limiting the availability of evidence-based findings. This study compared the outcomes of classification by dental students, orthodontic residents, and orthodontists. METHODS: Three different groups of students, orthodontic residents, and orthodontists evaluated 10 randomly chosen CBCT scans regarding midpalatal suture maturation from a pool of 179 patients (98 female and 81 male patients) aged 8 - 40 years which were previously classified by evaluating CBCT scans. The pool was set as benchmark utilizing midpalatal suture maturation classification by one examiner (OsiriX Lite version 11.0; Pixmeo SARL, Bernex, Switzerland). For assessment of intra-rater reliability of the examiners of each group the randomly chosen subjects were reclassified for midpalatal suture maturation after a wash-out period of two weeks by using the same software. Statistical analysis was performed to evaluate intra- and interrater reliability of the three groups with differing experience level. RESULTS: Groupwise intra-rater reliability assessment between the classification and reclassification was weak for examiners with a low level of experience (k = 0.59). Orthodontists had highest degree of agreement with regard to benchmark classification with an inter-rater reliability to be considered as moderate (k = 0.68). CONCLUSIONS: Assessment of midpalatal suture maturation on CBCT scans appears to be a subjective process and is considerably related to the experience level of the examiner. A high level of clinical experience seems to be favorable but does not necessarily ensure accurate results.

Age Estimation by Machine Learning and CT-Multiplanar Reformation of Cranial Sutures in Northern Chinese Han Adults.

OBJECTIVES: To establish age estimation models of northern Chinese Han adults using cranial suture images obtained by CT and multiplanar reformation (MPR), and to explore the applicability of cranial suture closure rule in age estimation of northern Chinese Han population. METHODS: The head CT samples of 132 northern Chinese Han adults aged 29-80 years were retrospectively collected. Volume reconstruction (VR) and MPR were performed on the skull, and 160 cranial suture tomography images were generated for each sample. Then the MPR images of cranial sutures were scored according to the closure grading criteria, and the mean closure grades of sagittal suture, coronal sutures (both left and right) and lambdoid sutures (both left and right) were calculated respectively. Finally taking the above grades as independent variables, the linear regression model and four machine learning models for age estimation (gradient boosting regression, support vector regression, decision tree regression and Bayesian ridge regression) were established for northern Chinese Han adults age estimation. The accuracy of each model was evaluated. RESULTS: Each cranial suture closure grade was positively correlated with age and the correlation of sagittal suture was the highest. All four machine learning models had higher age estimation accuracy than linear regression model. The support vector regression model had the highest accuracy among the machine learning models with a mean absolute error of 9.542 years. CONCLUSIONS: The combination of skull CT-MPR and machine learning model can be used for age estimation in northern Chinese Han adults, but it is still necessary to combine with other adult age estimation indicators in forensic practice.

Pathogenic variants in the paired-related homeobox 1 gene (PRRX1) cause craniosynostosis with incomplete penetrance.

PURPOSE: Studies have previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the preosteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function (LoF) variants in PRRX1 associated with craniosynostosis. METHODS: Trio-based genome, exome, or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins. RESULTS: Genome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis, who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9 of 1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, 7 additional individuals (4 families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multisuture synostosis was present in 11 of 17 cases (65%). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis. CONCLUSION: This work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis.

Synchondrosis fusion contributes to the progression of postnatal craniofacial dysmorphology in syndromic craniosynostosis.

Syndromic craniosynostosis (CS) patients exhibit early, bony fusion of calvarial sutures and cranial synchondroses, resulting in craniofacial dysmorphology. In this study, we chronologically evaluated skull morphology change after abnormal fusion of the sutures and synchondroses in mouse models of syndromic CS for further understanding of the disease. We found fusion of the inter-sphenoid synchondrosis (ISS) in Apert syndrome model mice (Fgfr2S252W/+ ) around 3 weeks old as seen in Crouzon syndrome model mice (Fgfr2cC342Y/+ ). We then examined ontogenic trajectories of CS mouse models after 3 weeks of age using geometric morphometrics analyses. Antero-ventral growth of the face was affected in Fgfr2S252W/+ and Fgfr2cC342Y/+ mice, while Saethre-Chotzen syndrome model mice (Twist1+/- ) did not show the ISS fusion and exhibited a similar growth pattern to that of control littermates. Further analysis revealed that the coronal suture synostosis in the CS mouse models induces only the brachycephalic phenotype as a shared morphological feature. Although previous studies suggest that the fusion of the facial sutures during neonatal period is associated with midface hypoplasia, the present study suggests that the progressive postnatal fusion of the cranial synchondrosis also contributes to craniofacial dysmorphology in mouse models of syndromic CS. These morphological trajectories increase our understanding of the progression of syndromic CS skull growth.